Bloom syndrome (BS) is a rare human genetic disease in which patients exhibit growth retardation, immunodeficiency, infertility, photosensitivity, and predisposition to cancer. The gene defective in BS (named BLM) was found to belong to an evolutionarily conserved RecQ helicase family. BLM gene belongs to the same helicase family as do the genes mutated in the premature aging diseases, Werner Syndrome (WS) and Rothmund-Thomson syndrome (RTS). All three diseases have some common features, notably genetic instability and predisposition to cancer. But each disease also has its own distinctive symptoms. To help understand the mechanism of these human diseases and their potential relevance to normal aging, we are isolating the protein complexes containing each gene product and characterized their functions. The results for BLM complexes are summarized below. We isolated from human HeLa extracts three complexes containing the BLM helicase. Interestingly, one of the complexes, termed BRAFT, also contained five Fanconi anemia (FA) complementation group proteins (FA) that were known at the time. FA resembles BS in genomic instability and cancer predisposition. FA patients also display a range of tissue-specific premature onset and accelerated aging (See project report on Fanconi anemia; ZO1 AG000688-07). FA gene products were not known to possess biochemical activity when these complexes were first isolated (2003), and the molecular pathogenesis of the disease was poorly understood. We demonstrated that BRAFT displays a DNA-unwinding activity that requires the presence of BLM, so that complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIa and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. Our study provided the first biochemical characterization of a multiprotein FA complex and suggested a connection between the BLM and FA pathways of genome maintenance. We identified RMI1 (also known as BLAP75) as a component of all three BLM complexes from HeLa cells. Using siRNA knockdown techniques, we showed that RMI1 is essential for BLM complex stability in vivo. Consistent with a role in BLM-mediated processes, RMI1 co-localized with BLM in subnuclear foci in response to DNA damage, and its depletion impaired the recruitment of BLM to these foci. Moreover, cells depleted of RMI1 displayed an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, RMI1 is an essential component of the BLM-associated cellular machinery that maintains genome integrity. It was shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase III (hTOPO III ), can process a toxic DNA intermediate generated in DNA repair reactions into a non-toxic product by a mechanism termed dissolution. In a collaboration with I. Hickson, G. Brown, and L. Lis labs, we found that RMI1 can strongly promote the dissolution catalyzed by hTOPO III by recruiting this enzyme to the toxic intermediate. This study demonstrates that BLM, hTOPO III and RMI1 function as a molecular machine that protects genome stability by efficiently processing toxic intermediates generated during DNA repair. We have collaborated with Lei Li's lab and inactivated RMI1 in mice. We found that RMI1 attenuates tumor development and is essential for mouse early embryonic development. We discovered a new component of the BLM complex, RMI2. RMI2 interacts with RMI1 (BLAP75) through two OB-fold domains similar to those in RPA, a single-strand DNA binding protein essential for replication and repair. The resulting complex, named RMI, differs from RPA in that it lacks obvious DNA binding activity. Nevertheless, RMI stimulates the dissolution of a homologous recombination intermediate in vitro and is essential for the stability, localization, and function of the BLM complex in vivo. Notably, inactivation of RMI2 in chicken DT40 cells results in an increased level of sister-chromatid exchange (SCE)--the hallmark feature of Bloom syndrome cells. Epistasis analysis revealed that RMI2 and BLM suppress SCE within the same pathway. Our data suggest that multi-OB-fold complexes mediate two modes of BLM action: one via RPA-mediated protein-DNA interaction and the other via RMI-mediated protein-protein interactions. We have since collaborated with two structural groups to solve the crystal structure of the RMI1-RMI2 core complex. The overall structure strongly resembles two-thirds of the trimerization core of RPA. Immunoprecipitation experiments with RMI2 variants confirm key interactions that stabilize the RMI core interface. Disruption of this interface leads to a dramatic increase in cellular sister chromatid exchange events similar to that seen in BLM-deficient cells. The RMI core interface is therefore crucial for BLM dissolvasome assembly and may have additional cellular roles as a docking hub for other proteins. Based on the crystal structure of RMI1, we also identified a region critical in mediating the interactions between RMI1 and Top3a. The RMI1-RMI2 structures therefore provide important molecular details of how the BLM complex is assembled. In collaboration with James Kecks group, we recently found that the RMI1-RMI2 complex directly interacts with FANCM, a DNA remodeling enzyme defective in some cases of Fanconi anemia. The RMI1-RMI2-complex serves as a bridge connecting BLM with FANCM. We have solved the crystal structure of the interface between RMI1-RMI2 and FANCM and identified key residues at the interface. Mutations of several residues at the interface disrupt the association between RMI and FANCM in vitro, and result in higher levels of sister-chromatid exchange, a hallmark feature of cells lacking BLM or FANCM. These data suggest that the interactions between RMI and FANCM are important for maintaining genome stability. In bioinformatics searches of the human genome, we noticed that human genome contains proteins with OB-fold domains similar to those in RMI and RPA. Two of them are single-strand DNA binding proteins, hSSB1 and hSSB2. We immunoprecipitated complexes containing both hSSB1 and hSSB2. We found that these two proteins form two separate complexes. Interestingly, both complexes contain two identical components, named INTS3 and SSBIP1. We have performed siRNA depletion, and found that cells depleted of these proteins have reduced efficiency of homologous recombination-dependent repair of DSB, sensitivity to DNA damaging agents, and deficieny in ATM-dependent signaling. Our data demonstrate that these two new complexes play important roles in protecting genome integrity in human. We have also identified Rif1 as a novel component of the BLM complex. We found that Rif1 works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA binding domain that resembles the CTD domain of bacterial RNA polymerase; and this domain preferentially binds fork and HJ DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA binding interface for the BLM complex to restart stalled replication forks.